Sarcoplasmic reticulum (SR) Ca release represents a large charge translocation that could very quickly (<1 ms) move the SR membrane potential (Vm) to the Ca equilibrium potential (ECa), where net Ca release will cease (1). However, this does not happen because there is a simultaneous countercurrent during release. In the previous funded period, we established that the multi-ion, poorly selective, pore of the ryanodine receptor (RyR) mediates large counter Mg and K fluxes, while it releases Ca. The RyR therefore carries the bulk of required countercurrent itself. This leaves the physiological roles of the SR K and Cl channels unclear. The SR K and Cl channels may carry a small important element of countercurrent during release, but they may also provide vital pathways for counter ions to re-equilibrate across the SR after release. Indeed, the mechanisms of SR ion and charge balance are so poorly understood that we do not know their significance to SR Ca handling, their possible pathological contributions or whether they can be exploited for therapeutic benefit. In 2010, the trimeric intracellular cation (TRIC) protein was identified as the SR K channel (2). Ablation of both TRIC isoforms (A & B) is embryonically lethal. Ablation of just TRIC-A (predominant form in skeletal muscle) generates clear in SR Ca handling abnormalities (abnormal local Ca release events and SR Ca overload; (3-5)). TRIC-A KO muscle not only proves there is a mechanistic link between SR Ca handling and SR ion balance, but provides a unique opportunity to define it. Here, we will use TRIC-A KO muscle to define how the complex spatiotemporal Ca, Mg, K and Cl fluxes through RyR, SR K and SR Cl channels control SR ion (voltage) balance and modulate SR Ca handling in skeletal muscle. The hypothesis tested here is: The SR K (TRIC) and SR Cl channels do not carry essential countercurrent during individual skeletal muscle SR Ca release events but instead provides crucial SR ion re-equilibration pathways, assuring SR ion balance and that SR Vm returns to resting values between release events. Defective re-equilibration, not missing countercurrent, explains the SR Ca handling defects observed in TRIC-KO skeletal muscle. This is tested by the following specific aims. Aim 1: Determine if SR K or Cl channels carry essential countercurrent during SR Ca release. Aim 2: Determine if SR K or Cl channels provide vital resting SR ion re-equilibration pathways. Aim 3: Establish the mechanism underlying abnormal SR Ca handling in TRIC-A KO muscle. Expected Outcome: Delineate new potential points of SR pathological failure (and/or sites at which SR function can be therapeutically manipulated) by transforming existing Ca-centric view of SR function to encompass the multi-ion (Ca, Mg, K, Cl) reality, which governs SR Ca release/uptake.